There are several alternatives for the production of eukaryotic proteins in different expression systems. The following expression systems are currently in use.
Bacteria
Many E. coli expression systems are commercially available. Some examples are pET (Promega), pQE (Qiagen), pGEX (Amersham Pharmacia), ptrcHIS (Invitrogen), pDUAL (Stratagene). The advantage of E. coli systems are that they are cheap and very easy to use. The main disadvantage is that many eukaryotic proteins do not fold properly when expressed in E. coli and form insoluble aggregates. Codon usage is very different from that in higher eukaryotes. Often eukaryotic proteins must be modified following translation in order to be able to fold into the proper structure and/or to become activated. E. coli is not able to carry out complex post translational modifications such as acetylation, N- and O-linked glycosylation and, acylation and phosphorylation which are exclusively performed by eukaryotic cells.
The levels of expression vary enormously from protein to protein. The yield of recombinant protein from 1 liter of E. coli culture amounts typically to about 10 mg. In rare cases amounts of hundreds of milligrams of recombinant protein per liter E. coli culture can be obtained.
Yeast
The yeast Pichia pastoris is a well established system for expressing recombinant proteins. Several companies sell the relevant plasmid vectors. Invitrogen Corp. for example sells the pPIC set of plasmids. The advantages of Pichia is that being a eukaryote, the post-translational modifications are more similar to those that occur in humans or higher eukaryotes. Pichia is easy and fast to transform. It is also easy to grow on a large scale. Expression levels vary considerably. Levels as high as 12 grams/liter have been reported.
Insect Cells
Insect cells can also be used to express recombinant proteins. Several companies sell the relevant plasmid vectors and cell lines. Invitrogen's systems are DES, InsectSelect and MaxBac. The advantages of insect cells are that, being multicellular eukayotes, insects are much more like humans than yeast are. The disadvantages are (i) insect cells are much more difficult to grow and maintain than bacterial or yeast cells (ii) they are more expensive to cultivate (iii) they require sterile incubators (iv) the expression levels are much lower than those seen with the yeast or bacterial systems.
Human Cell Lines
Human cell lines can also be used, but they require growth factors in order to keep them alive. Today, human growth factors are very expensive, and this makes human cell lines bad candidates for e.g. growth factor production.
Nematodes
Nematodes, small roundworms, are one branch of eukaryotic organisms that have so far not been exploited for large-scale protein or nucleic acid production. Nematodes are very simple animals and have served as a developmental model system ever since 1949 (Dougherty E C and Nigon V., J. Parasitol. (1949) 35, 11; Brenner S in a letter to Max Perutz, 5 Jun. 1963). Especially the development of each of the 959 cells in the nematode C. elegans is well characterized and its entire genome was recently sequenced and is now publicly available (“The C. elegans Sequencing Consortium” Genome Sequence of the nematode C. elegans: A platform for investigating biology. Science (1998) 282, 2012-2018). Nematodes are eukaryotic organisms that are genetically much more closely related to humans than bacteria, about 60% of their proteins are homologous to human proteins.
What makes nematodes interesting as a protein expression system for production of eukaryotic and in particular human proteins is the fact that they are equipped with the necessary machinery to perform post-translational modifications on proteins. Hence, proteins (peptide drugs) produced in nematodes are virtually identical to the natural human proteins and as a result may have fewer undesirable side effects and a higher specific activity requiring lower dosages. Other advantages of the nematode expression system include high yields of expressed protein, its low maintenance costs, its ease of use and that one can easily scale up the production.
Expression of human beta-amyloid peptide in transgenic C. elegans to produce muscle-specific deposits immunoreactive with anti-beta-amyloid polyclonal and monoclonal antibodies has been described by C. D. Link (Link C. D., Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans, Proc. Natl. Acad. Sci. (1 995) 92,9368-9372), and he suggests that his invertebrate model may be useful for in vivo investigation of factors that modulate amyloid formation.
The international patent application WO 00/54815 discloses expression of DNA or proteins in C. elegans by using an expression vector comprising a promoter that directs the gene expression to the excretory cell of C. elegans. The reason for the protein expression is not production and isolation of a protein but for discovery of novel molecules, i.e. drugs, involved in the cell motility, cell shape and cell outgrowth process, and to establish their function.